1. Volume 54, Issue 5, Pages 737-750 (June 2014)
A DDX6-CNOT1 Complex and W-Binding Pockets in CNOT9 Reveal Direct Links between miRNA Target Recognition and Silencing 
Ying Chen, Andreas Boland, Duygu Kuzuoğlu-Öztürk, Praveen Bawankar, Belinda Loh, Chung-Te Chang, Oliver Weichenrieder, Elisa Izaurralde 
Molecular Cell 
Volume 54, Issue 5, Pages (June 2014)
DOI: /j.molcel
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2. Molecular Cell 2014 54, 737-750DOI: (10.1016/j.molcel.2014.03.034)
Copyright © 2014 Elsevier Inc. Terms and Conditions

3. Figure 1
Structure of the CNOT1 CN9BD Bound to the CNOT9 ARM Repeat Domain
(A) Diagram of CNOT1 with N-terminal, middle, and C-terminal regions (CNOT1-N, CNOT1-M, and CNOT1-C, respectively). CNOT1-N consists of two HEAT-like repeat domains. CNOT1-M contains an MIF4G domain and the CN9BD (previously DUF3819). CNOT1-C contains the NOT1 superfamily homology domain (SHD). CNOT9 contains an armadillo (ARM) repeat domain. TNRC6C contains and N-terminal AGO-binding domain (ABD), a ubiquitin-associated-like domain (UBA), and a C-terminal silencing domain (SD). The SD comprises a Mid region, an RNA recognition motif (RRM), and a C-terminal (C-term) region. The positions of the CCR4-NOT interacting motifs 1 and 2 (CIM-1 and CIM-2) and the PAM2 motif (PABP-interacting motif 2) are indicated. Vertical green and red lines indicate the positions of W-containing motifs binding to AGOs and deadenylases, respectively. Amino acid positions at domain boundaries are indicated below the protein outlines. See also Figure S1A.
(B and C) Western blot showing the interaction between GFP-CNOT1 fragments and HA-MBP-tagged CNOT9 and HA-tagged TNRC6A-SD (6A-SD) or TNRC6C-SD (6C-SD) in HEK293T cells. GFP-F-Luc served as a negative control. See also Figures S1B–S1H.
(D and E) Overall structure of the CN9BD-CNOT9 binary complex in two orientations. CNOT9 is shown in cyan; the three long α helices in the CNOT1 CN9BD are shown in pink, yellow, and green. Bound W residues are shown as magenta sticks. Secondary structure elements are labeled in blue for CNOT9 and in black for CNOT1.
(F) Cartoon representation of the ARM repeat domain of CNOT9. The ARM repeats are colored in a gradient from pink to dark blue from the N to C terminus.
(G) Surface representation of the CN9BD-CNOT9 binary complex in the same orientation as that in (D) and colored according to surface potential contoured from −5 kT/e (red) to +5 kT/e (blue). The position of the DNA/RNA binding surface is indicated. See also Figures S1F and S1G.
Molecular Cell  , DOI: ( /j.molcel )
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4. Figure 2 The CN9BD-CNOT9 Binding Interface
(A–D) Overview and close-up views of the interface between the CNOT1 CN9BD and the CNOT9 ARM domain, with selected interface residues shown as sticks. Residues mutated in this study are underlined and shown as red sticks (CNOT1) or dark blue sticks (CNOT9). The small and large rectangles in (A) indicate the views shown in (C) and (D), respectively. Residues and secondary structure elements are labeled in black for CNOT1 and in blue for CNOT9. Hydrogen bonds are shown as red dashed lines. See also Figures S2–S4.
(E) Interaction of GFP-CNOT9 (full-length wild-type or quadruple mutant, Mut1) with HA-CNOT1 (full length) in human cells. See also Figure S4.
(F) Interaction of GFP-CNOT1 (full-length wild-type or mutants) with CNOT9-HA-MBP in HEK293T cells.
(G) Interaction of GFP-tagged Dm NOT9 (wild-type or mutants) with HA-NOT1 in Dm S2 cells.
(H) Interaction of GFP-tagged Dm NOT9 with HA-NOT1 (wild-type or mutants) in Dm S2 cells. In all panels, cell lysates were treated with RNase A prior to immunoprecipitation. See also Figure S4 and Table S1.
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5. Figure 3 CNOT9 Harbors Tandem W-Binding Pockets
(A) Overview of the CN9BD-CNOT9 complex is shown for orientation. The W residues (W1 and W2 corresponding to CNOT1 W1603 and free W, respectively) bound to CNOT9 are shown as sticks.
(B) Close-up views of the W-binding pockets. The right panel shows a surface representation of the view in the left panel, with the CNOT9 surface colored white to yellow with increasing hydrophobicity (scores according to Kawashima et al., 2008).
(C and D) Close-up views of W-binding pocket 1 (C) (high-resolution structure) and W-binding pocket 2 (D) (low-resolution structure). The electron difference densities (Fo-Fc, contoured at 2.5 σ) for the W-containing peptide and the free W residue are shown as a gray mesh, and the corresponding structural models are displayed as magenta sticks. Residues mutated in this study are underlined. Hydrogen bonds are shown as red dashed lines. See also Figures S2 and S4 and Table S1.
Molecular Cell  , DOI: ( /j.molcel )
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6. Figure 4 The CNOT9 W-Binding Pockets Mediate Binding to the TNRC6s
(A) MBP pull-downs using recombinant MBP-tagged 6A-SD and His-tagged CNOT9 ARM domain (wild-type or the indicated mutants). MBP served as a negative control. See also Figure S5.
(B) MBP pull-downs using recombinant MBP-tagged 6A-SD and His-tagged CNOT9 ARM domain (wild-type or the indicated mutants).
(C) Interaction between GFP-CNOT9 (wild-type or the indicated mutants) and HA-6A-SD in the presence of HA-MBP-tagged CN9BD in HEK293T cells.
(D) Interaction between GFP-CNOT9 (wild-type or the indicated mutants) and full-length HA-CNOT1 in HEK293T cells.
(E) Interaction between GFP-CNOT1 (wild-type or the 4×M and 5×M mutants that do not bind CNOT9) and HA-6A-SD in HEK293T cells.
(F) Interaction between GFP-CNOT9 (wild-type or the indicated mutants) and HA-GW182 in the presence of HA-tagged CN9BD in Dm S2 cells. See also Figure S5 and Table S1.
(G) HeLa cells (transfected with a control shRNA) or cells depleted of CNOT1 were transfected with a mixture of three plasmids: the psiCHECK-8×Let-7 or the corresponding reporter carrying mutations in Let-7-binding sites (R-Luc-Mut), a plasmid expressing F-Luc as a transfection control, and a plasmid expressing shRNA-resistant versions of GFP-CNOT1 (wild-type or 5×M mutant) or GFP. For each condition, Renilla luciferase activity was measured, normalized to that of the F-Luc transfection control, and set at 100% in cells expressing R-Luc-Mut (black bars). Mean values ± SD from five independent experiments are shown.
(H) Western blots showing the efficiency of the CNOT1 knockdown and the expression levels of endogenous CNOT9. Dilutions of control cell lysates were loaded in lanes 1–4 to estimate the efficacy of the depletion. α-tubulin served as a loading control. The asterisk indicates the position of the GFP-CNOT1 used in the complementation assay. See also Figures S5N and S5O.
Molecular Cell  , DOI: ( /j.molcel )
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7. Figure 5
Structure of the CNOT1 MIF4G Domain Bound to the DDX6 RecA-C Domain
(A) DDX6 consists of two RecA-like domains, termed RecA-N and RecA-C, connected by a flexible linker.
(B–D) Interaction of GFP-CNOT1 or GFP-eIF4G with HA-tagged DDX6 (B), eIF4A1 (C), or eIF4A2 (D) in HEK293T cells. See also Figure S6.
(E and F) Overall structure of the CNOT1 MIF4G-DDX6 RecA-C complex (this study) (E) and the Saccharomyces cerevisiae eIF4G MIF4G-eIF4A complex (2VSO; Schütz et al., 2008) (F). Selected secondary structure elements are indicated.
(G and H) Close-up views of the interface between the CNOT1 MIF4G domain and the DDX6 RecA-C domain showing the DDX6 arginine anchor residue R375 (G) and loop L3 (H). Selected interface residues are shown as sticks and colored green (CNOT1) or orange (DDX6). Residues and secondary structural elements are labeled in green for CNOT1 and in black for DDX6. Residues mutated in this study are underlined.
(I) Superposition of Sc eIF4A loop L3 (residues 255–262) onto Hs DDX6 L3 (329–336). Selected interface residues in loop L3 of the DDX6 and eIF4A RecA-C domains are shown as sticks and colored in orange (DDX6) and gray (eIF4A). The CNOT1 MIF4G residues that specifically form hydrogen bonds with DDX6 residues are shown in green. The residues that contribute to the specificity of the interaction are underlined and shown in bold. eIF4A residues are labeled in italics. Backbone cartoons of Sc eIF4G and eIF4A are omitted for clarity.
(J) A structural model built by superposition of DDX6 bound to the EDC3 FDF peptide (PDB 2WAX), DDX6 bound to the CNOT1 MIF4G domain (this study), and the CNOT1 MIF4G bound to CAF1 (PDB 4GMJ). The RNA is modeled based on the structure of Vasa bound to RNA (PDB 2DB3). See also Figure S6J.
Molecular Cell  , DOI: ( /j.molcel )
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8. Figure 6 Validation of the CNOT1 MIF4G-DDX6 Binding Interface
(A) Interaction between HA-DDX6 (wild-type or the indicated mutants) and GFP-CNOT1 in HEK293T cells. GFP-MBP served as negative control.
(B) MBP pull-downs using recombinant MBP-tagged CNOT1 MIF4G domain and His-tagged DDX6 RecA-C (wild-type or the R375A mutant). MBP served as a negative control.
(C) Interaction between GFP-CNOT1 (wild-type or the indicated mutants) and HA-DDX6 in HEK293T cells. See also Figure S6 and Table S1.
(D) Western blots showing the efficiency of the DDX6 knockdown. Dilutions of control cell lysates were loaded in lanes 1–4 to estimate the efficacy of the depletion. α-tubulin served as a loading control. The asterisk indicates the position of the HA-DDX6 used in the complementation assay shown in (E).
(E) A complementation assay was performed as described in Figure 4G in cells depleted of DDX6. Mean values ± SD from three independent experiments are shown. See also Figures S5N, S6E, and S6H.
(F) Western blot analysis showing the equivalent expression of the DDX6 proteins used in the complementation assay.
Molecular Cell  , DOI: ( /j.molcel )
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